This proposal is concerned with: 1) how actin is maintained in an insoluble, yet unpolymerized form (profilactin) in echinoderm sperm, 2) what controls and what is the packing of actin filaments in Limulus sperm, in stereocilia of the ear, in gut microvilli, and in the cnidocil, 3) how are the length and polarity of actin filaments controlled, and 4) how do fluxes of ions into the cell control the actin antics in eggs and sperm. The five proteins making up the profilactin will be isolated and characterized, and their role in inhibiting actin polymerization and/or in making the actin insoluble will be determined using NBD chloride actin. The role that nucleotides play in polymerization in vitro and in vivo will be assayed. I will also assay the profilactin for bound lipid. I propose to compare the chemical composition of the filament bundle in Limulus sperm in its three states, the coil, the false discharge, and the true discharge so that we can see what changes occur in the bound proteins when there is a change in pitch of the actin helix. We will also try to locate the position of the 95,000 molecular weight protein. A detailed study in collaboration with David DeRosier using optical diffraction and 3d reconstruction techniques on the actin filament bundles in the stereocilia of the ear, on the filament bundles in microvilli, and on filament bundles in the cnidocil will be carried out. We propose to use fast freezing, freeze fracturing, and etching techniques, and to study embryonic ear stereocilia. I will also try to determine what ions must enter the cell locally to allow polymerization of actin in eggs and sperm. I propose to use microinjection techniques. I will attempt to see how the filament bundles become ordered in fertilization cones and cytokinesis.